Methods for detecting multiple target nucleic acids in multiple samples by use nucleotide tags
The present invention provides assay methods that increase the number of samples and/or target nucleic acids that can be analyzed in a single assay. In certain embodiments, an assay method entails separately subjecting S samples to an encoding reaction that produces a set of T tagged target nucleotide sequences, each tagged target nucleotide sequence including a sample-specific nucleotide tag and a target nucleotide sequence. In some embodiments, an assay method entails separately subjecting S samples to an encoding reaction that produces a set of T tagged target nucleotide sequences, each tagged target nucleotide sequence including a first nucleotide tag linked to a target nucleotide sequence, which is linked to a second nucleotide tag. In either case, the tagged target nucleotide sequences from the S samples can be mixed to form an assay mixture and subsequently assayed.
1. An assay method for detecting a plurality of target nucleic acids in a plurality of samples, the method comprising:
providing S samples that will be mixed together prior to assay, where S is an integer greater than 1;
separately subjecting each of said S samples to an encoding reaction that produces a set of T tagged target nucleotide sequences, each tagged target nucleotide sequence comprising a sample-specific nucleotide tag and a target nucleotide sequence; wherein T is the number of target nucleic acids to be detected, T being an integer greater than one;
mixing together tagged target nucleotide sequences from said S samples to form an assay mixture;
dividing the assay mixture into S×T amplification mixtures, wherein S×T is at least 30, and separately and simultaneously subjecting each of said amplification mixtures to amplification using a unique pair of amplification primers, wherein each amplification primer pair comprises:
a forward or a reverse amplification primer that anneals to a target nucleotide sequence; and
a reverse or a forward amplification primer, respectively, that anneals to a sample-specific nucleotide tag; and
for each unique primer pair, determining whether an amplification product is present in the amplification mixture; and
if the amplification product is present, determining that a particular target nucleic acid is present in a particular sample; or
if the amplification product is not present, determining that the particular target nucleic acid is not present in the particular sample.
2. The method of claim 1 , wherein:
said encoding reaction comprises separately subjecting each of said S samples to preamplification using a distinct set of forward and reverse preamplification primers for each sample to produce preamplified samples, wherein
each preamplification primer set comprises T pairs of forward and reverse preamplification primers, wherein each preamplification primer pair is capable of amplifying a particular target nucleic acid; and
either all forward preamplification primers or all reverse preamplification primers in a given set comprise a common sample-specific nucleotide tag; and
said mixing comprises mixing the preamplified samples for each of said S samples to form an assay mixture.
3. The assay method of claim 2 , wherein each forward preamplification primer in a set comprises a common sample-specific nucleotide tag, in addition to a target-specific nucleotide sequence, and each reverse preamplification primer in each set comprises a target-specific nucleotide sequence.
4. The assay method of claim 2 , wherein each forward preamplification primer in a set comprises a target-specific nucleotide sequence, and each reverse preamplification primer in each set comprises a common sample-specific nucleotide tag, in addition to a target-specific nucleotide sequence.
5. An assay method for detecting a plurality of target nucleic acids in a plurality of samples, the method comprising:
providing S samples that will be mixed together prior to assay, where S is an integer greater than 1;
separately subjecting each of said S samples to an encoding reaction that produces a set of T tagged target nucleotide sequences, each tagged target nucleotide sequence comprising a first nucleotide tag linked to a target nucleotide sequence, which is linked to a second nucleotide tag; wherein T is the number of target nucleic acids to be detected, T being an integer greater than one;
mixing together tagged target nucleotide sequences from said S samples to form an assay mixture;
dividing the assay mixture into S×T amplification mixtures, wherein S×T is at least 30, and separately and simultaneously subjecting each of said amplification mixtures to amplification using a unique pair of amplification primers, wherein each amplification primer pair comprises:
a forward or a reverse amplification primer that anneals to a first nucleotide tag; and
a reverse or a forward amplification primer, respectively, that anneals to a second nucleotide tag; and
for each unique primer pair, determining whether an amplification product is present in the amplification mixture; and
if the amplification product is present, determining that a particular target nucleic acid is present in a particular sample; or
if the amplification product is not present, determining that the particular target nucleic acid is not present in the particular sample.
6. The method of claim 5 , wherein:
said encoding reaction comprises separately subjecting each of said S samples to preamplification using a distinct set of forward and reverse preamplification primers for each sample to produce preamplified samples, wherein
each preamplification primer set comprises T pairs of forward and reverse preamplification primers, wherein each preamplification primer pair is capable of amplifying a particular target nucleic acid; and
each forward preamplification primer comprises a forward nucleotide tag, and each reverse preamplification primer comprises a reverse nucleotide tag; and
said mixing comprises mixing the preamplified samples for each of said S samples to form an assay mixture.
7. The method of claim 5 , wherein at least one of said nucleotide tags comprises a sample-specific nucleotide tag that is common to all tagged target nucleotide sequences produced from a given sample.
8. The method of claim 7 , wherein the other nucleotide tag is distinct for each tagged target nucleotide sequence in the assay mixture.
9. The assay method of claim 1 or 5 , wherein at least one of said amplification primers, comprises at least one nucleotide that is complementary to the target nucleotide adjacent to at least one of said nucleotide tags.
10. The assay method of claim 1 or 5 , wherein a series of samples is assayed by preparing a plurality of different assay mixtures, wherein each assay mixture comprises a mixture of S different samples.
11. The assay method of claim 10 , wherein S×T is at least a value selected from the group consisting of 48, 96, 120, and 192.
12. The assay method of claim 10 , wherein the product of the total number of samples assayed in a single assay ×T is at least a value selected from the group consisting of 2304, 3600, 4608, and 9216.
13. The assay method of claim 2 or 6 , wherein amplification mixtures are formed in or, distributed into, separate compartments of a microfluidic device prior to amplification.
14. The assay method of claim 13 , wherein the microfluidic device is fabricated, at least in part, from an elastomeric material.
15. The assay method of claim 13 , wherein the assay has a dynamic range of at least 4 orders of magnitude.
16. The assay method of claim 2 , wherein the preamplification and/or the amplification is carried out by polymerase chain reaction (PCR).
17. The assay method of claim 2 or 6 , wherein the preamplification is carried out for 2-20 cycles to introduce the nucleotide tags.
18. The assay method of claim 2 or 6 , wherein the preamplification is carried out for a sufficient number of cycles to normalize amplicon copy number across targets and across samples.
19. The assay method of claim 2 , wherein the presence of an amplification product is determined by quantitative real-time polymerase chain reaction (qPCR).
20. The assay method of claim 2 , wherein a universal qPCR probe is employed in the amplification mixtures to detect amplification products.
21. The assay method of claim 2 , wherein one or more target-specific qPCR probes is employed in the amplification mixtures to detect amplification products.
22. The assay method of claim 2 or 6 , wherein one or more tag-specific qPCR probes is employed in the amplification mixtures to detect amplification products.
23. The assay method of claim 2 , wherein the presence of an amplification product is detected using a fluorogenic nuclease assay.
24. The assay method of claim 23 , wherein the presence of an amplification product is detected using a dual-labeled fluorogenic oligonucleotide probe.
25. The assay method of claim 2 , additionally comprising quantifying the amount of amplification product in the amplification mixtures.
26. The assay method of claim 25 , additionally comprising determining the amount of each target nucleic acid present in each sample.
27. The assay method of claim 2 , wherein the assay is performed to determine the copy numbers of the target nucleic acids.
28. The assay method of claim 2 , wherein the assay is performed to determine genotypes at loci corresponding to the target nucleic acids.
29. The assay method of claim 2 , wherein the assay is performed to determine the expression levels of the target nucleic acids.
30. The assay method of claim 2 , additionally comprising, reducing the concentration of preamplification primers prior to carrying out said amplification.
31. The method of claim 2 or 6 , wherein the sample comprises a genomic DNA sample.
32. The method of claim 31 , wherein the preamplification is conducted in the presence of an amount of a blocking agent that is sufficient to increase specific amplification of the target nucleic acid.
33. The method of claim 32 , wherein the blocking agent comprises a nucleic acid blocking agent that hybridizes to repetitive sequences in the genomic DNA sample.
34. The method of claim 32 , wherein the blocking agent is selected from the group consisting of tRNA, degenerate oligonucleotide primers, repetitive DNA, bovine serum albumin (BSA), and glycogen.
35. The method of claim 32 , wherein the blocking agent is present at a concentration in the range of about 0.1 μg/μl to about 40 μg/μl.
36. The method of claim 35 , wherein the blocking agent comprises tRNA at a concentration in the range of about 1 μg/μl to about 5 μg/μl.